Combinatorial approaches to protein folding and function

描述：(申请人提供)蛋白质的折叠和稳定性 在癌症等疾病的分子理解中成为关键要素 和阿尔茨海默氏症？S，然而对这些现象的完整理解仍然 难以捉摸。一种卓有成效的方法是系统地重新设计 蛋白质。重新设计的一个重要限制是对 可以设计出数量巨大的可能的变种。的方法。 对研究良好的蛋白质的大型变异体文库的分析是非常重要的 令人向往。一个研究非常充分的蛋白质折叠和稳定性模型是 4-螺旋束蛋白Rop，它调节Cole 1质粒的拷贝数。 这将作为Rop功能的屏幕的基础，其中 利用Cole-1质粒生产类绿色荧光蛋白标记物 反映了Rop蛋白变体的完整性。这样的屏幕将用于 从包含详尽信息的大型库中识别Active Rop变体 环或核心残基的突变。一个有趣的问题是 识别控制整体构象的参数。近期 实验表明，蛋白质可以折叠成高度不同的三维结构 结构，尽管同源性很高(例如， α/βB1结构域折叠成Rop的卷曲线圈结构)。类ROP 通过新型的工程技术，金属离子稳定了结构 金属结合部位。我们将通过以下方式研究构象转换的限制 使用金属结合来稳定B1变异体的Rop-like结构。我们是 还对分离概括Rop功能的B1变体感兴趣。一个 Rop功能的筛选将使我们从根本上扩大蛋白质的范围 重新设计和完善我们对控制蛋白质的参数的理解 折叠和稳定。

DESCRIPTION: (provided by applicant) Folding and stability of proteins have emerged as key elements in the molecular understanding of diseases like cancer and Alzheimer?s, yet a complete understanding of these phenomena remains elusive. One highly productive approach has been the systematic redesign of proteins. A significant limitation to redesign is the characterization of the enormous number of possible variants that can be engineered. Methods for the analysis of large libraries of variants of well-studied proteins are highly desirable. An extremely well-studied model for protein folding and stability is the four-helix bundle protein Rop, which modulates ColE 1 plasmid copy number. This will serve as the basis for a screen for Rop function, wherein the production of a marker like green fluorescent protein from a ColE 1 plasmid reflects the integrity of a Rop protein variant. Such a screen will be used to identify active Rop variants from large libraries containing exhaustive mutations in loop or core residues. One interesting problem is the identification of parameters that control overall conformation. Recent experiments show that proteins can fold into highly disparate threedimensional structures despite high homology (for example, a variant of the alpha/beta B1 domain folds into the coiled-coil structure of Rop). Rop-like structures have been stabilized by metal ions through the engineering of novel metal-binding sites. We will examine the limits of conformational switching by using metal binding to stabilize a Rop-like structure from a B1 variant. We are also interested in isolating B1 variants that recapitulate Rop function. A screen for Rop function will allow us to radically expand the scope of protein redesign and refine our understanding of the parameters that control protein folding and stability.